1 nucleotide metabolism metabolism of pyrimidine nucleotides nucleotide metabolism metabolism of...
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NUCLEOTIDE METABOLISMNUCLEOTIDE METABOLISMMetabolism of pyrimidine nucleotidesMetabolism of pyrimidine nucleotides
NUCLEOTIDE METABOLISMNUCLEOTIDE METABOLISMMetabolism of pyrimidine nucleotidesMetabolism of pyrimidine nucleotides
ProfProf. Mária Sasvári. Mária Sasvári
Gergely Keszler2009.
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The structure of nucleobases
N-containing, heterocyclic aromatic compounds; substituted purine or pyrimidine rings
RNA DNA
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BLOOD
Pyrimidine nucleotidesPyrimidine nucleotidesPyrimidine nucleotidesPyrimidine nucleotides
cytidine
uridine
PRPP
“de novo” synthesis
CTP
UTP
Catabolism
CO2, NH3, -alanine
CoA
salvagereactions
CMP
UMP
dTTP
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UTP
UDP
UMP
CTP“de novo” synthesis
AspGlnCO2
CADPy 1-3
H2OAmito
Py 4
UMP-SPy 5,6 OA
PYRIMIDINE nucleotide synthesisPYRIMIDINE nucleotide synthesisPYRIMIDINE nucleotide synthesisPYRIMIDINE nucleotide synthesis
nucleosides
salvagereactions
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CO2
2.
Asp
““de novo” PYRIMIDINE synthesisde novo” PYRIMIDINE synthesis““de novo” PYRIMIDINE synthesisde novo” PYRIMIDINE synthesis
1.
Glu
Gln2 ATP
2 ADP + Pi
NH2
CO O - P
carbamoyl-P
Carbamoyl-Psynthetase II (CPSII)C
Py1
Asp transcarbamoylaseAPy2
AspGln
CADPy 1-3
H2OA
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AspGln
CADPy 1-3
H2OA““de novo” PYRIMIDINE synthesisde novo” PYRIMIDINE synthesis““de novo” PYRIMIDINE synthesisde novo” PYRIMIDINE synthesis
3.DihydroorotaseD
Py3
NH2
C
O NH
carbamoyl-Asp
COO-
COO -
HN
O NH
Dihydroorotate (H2OA)
COO-
O
H2O
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H2OAmito
Py 4OA ““de novo” PYRIMIDINE synthesisde novo” PYRIMIDINE synthesis““de novo” PYRIMIDINE synthesisde novo” PYRIMIDINE synthesis
Dihydroorotatedehydrogenase
Py4
HN
O NH
orotate (OA)
COO-
O
4.NADH + H+
NAD+
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UMP-SPy 5,6 OA
UMP
UMP-S(synthase)
Py5
Py6
““de novo” PYRIMIDINE synthesisde novo” PYRIMIDINE synthesis““de novo” PYRIMIDINE synthesisde novo” PYRIMIDINE synthesis
Orotatephosphoribosyl transferase 5.
PPi
PRPP
HN
O N COO-
O
R- POrotidylate (OMP)
UMP
6. CO2
OMP decarboxylase
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UTP
UDP
UMP7.
Pyrimidine monophosphatekinase
ADP
ATPUMP
UDP
8.Pyrimidine diphosphate
kinaseADP
ATP
UTP (2,4-dioxo)
CTP
9.CTP synthetase
Glu
Gln
CTP (2-oxo-4 amino)
ADP
ATP
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UMP
Regulation of Regulation of PYRIMIDINE “de novo” synthesisPYRIMIDINE “de novo” synthesis
Regulation of Regulation of PYRIMIDINE “de novo” synthesisPYRIMIDINE “de novo” synthesis
CADPy 1-3
CAD complex:a single polypeptidethree active centers+ allosteric sites
UTP
+ PRPP
UMP-SPy 5,6 OA
UMP
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Comparison of CPS I and II isoenzymes
CPSI CPSII
• role urea cycle de novo pyrimidine biosynthesis
• localization mitochondrial cytosolic
• N-donor free NH3 glutamine
• regulation + N-acetyl-glutamate + PRPP, - UTP
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Oral uridine
UTP
UDP
UMP
CTP
AspGln
CADPy 1-3
H2OAmito
Py 4
UMP-SPy 5,6 OA
Orotic aciduriaOrotic aciduriaOrotic aciduriaOrotic aciduria
1. UMP-S deficiency
2. Inhibition of UMP-S Allopurinol treatment (gout) 6-azauridine treatment (tumor)
3. Ornitine transcarbamoylase deficiency (urea cycle): NH3 CP Type II hyperammonemia, carbamoyl P (CP) accumulation
CAD can use the leaking CP orotic acid overproduction orotic aciduria
treatment: oral uridine
Main reasons of orotic aciduria
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citidin
uridin
CMP
UMP
SALVAGESALVAGENucleoNucleosside ide nucleo nucleottideide
SALVAGESALVAGENucleoNucleosside ide nucleo nucleottideide
Cytidylate deaminase
UMP
Br -p
Br
uridine cytidine
CMP
Uridine/cytidine kinase
H2ONH3
ATP
ADP
ATP
ADP
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Br -p
UMP (TMP) CMP
uridine (thymidine) cytidineB
r
Pi Pi
5’nucleotidase
cytidine deaminase
H2ONH3
CATABOLISMCATABOLISMNucleoNucleottide ide Nucleo Nucleossideide
CATABOLISMCATABOLISMNucleoNucleottide ide Nucleo Nucleossideidecytidine
uridine
CMP
UMP
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cytidine deaminase
pyrimidine nucleoside phosphorylase
dihydropyrimidine dehydrogenase
dihydropyrimidinase
ureidopropionase
Coenzyme A
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β-D-ribofuranose (in RNA) 2’-deoxy-β-D-ribofuranose (in DNA)
In cells, deoxyribonucleotides are synthesised from ribonucleotides by theribonucleotide reductase enzyme
Formation of deoxynucleotides
NADPH + H+ NADP+
H2O
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The ribonucleotide reductase reaction The ribonucleotide reductase reaction (RR)(RR)
The ribonucleotide reductase reaction The ribonucleotide reductase reaction (RR)(RR)
RR
S
SRR
SH
SH
TR
S
STR
SH
SH
FADH2 FAD
NDP dNDP
TR:thioredoxin reductase
or:glutaredoxin reductase
(glutathion)
NDP:UDP/ADP/GDP/CDP
NADPH+ H+ NADP+
H2O
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Mechanism of the RR reaction
ribonucleotide reductase
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The RR is a dimer
R1 – catalytic subunit constitutive expression
R2 – regulatory subunit induced in the S-phase
RR is active in dividing cells!
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Allosteric REGULATIONAllosteric REGULATIONAllosteric REGULATIONAllosteric REGULATION
1. dATP: complete inhibition
2. Regulation of the ratio
Substrates:
CDP
UDP
GDP
ADP
Products:
dCTP
dTTP
dGTP
dATP
+
+
dUDP dUMP
dCDP
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Synthesis of thymine nucleotides
Folate antagonists(chemotherapy!)
dTTP DNA
UMP
UDP
dUDP
dUDP
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5-FU, a potent inhibitor of thymidylate synthase
5-fluoro-uracil (5-FU)inhibitor of thymine synthesis – WHY??used to treat solid tumors (pancreas, lung, colon)
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Inhibition of the folate synthesis: antibacterial effectInhibition of the folate synthesis: antibacterial effectInhibition of the folate synthesis: antibacterial effectInhibition of the folate synthesis: antibacterial effect
Human: folic acid is a vitaminBacteria: synthesis of folate can be selectively inhibited
H2N S
O
O
N
H
R
sulphonamides
folic acid
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The role of folate and The role of folate and vitaminvitamin B B1212
in DNA synthesis and cell divisionin DNA synthesis and cell division
The role of folate and The role of folate and vitaminvitamin B B1212
in DNA synthesis and cell divisionin DNA synthesis and cell division
HomoCys
Met
THF
CH3-THF
CH2-THF
GlySer
dUMP dTMPpurine synthesis
Cell division
SAM
SAH-CH3
adenosine
B12
“methyl trap” B12/folate deficiency: pernicious anemia
oxidative stressatherosclerosis
folate cycleSAM/SAH
cycle
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Folate deficiency – megaloblastic anemia
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Vitamin B12 (cobalamin)deficiency
Methylmalonic aciduria Megaloblastic anemia(Pernicious anemia)
nerve degenerationparalysis and death
(B12 is needed for myelin synthesis)
Anemia is due to impaired dTTP synthesisfor DNA replication in hematopoetic precursor cells
might be due to achlorhydria (chronic gastritis, no IF production)treatment: parenteral administration of B12
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“ “Salvage” reaction of Salvage” reaction of deoxydeoxynucleosidesnucleosides “ “Salvage” reaction of Salvage” reaction of deoxydeoxynucleosidesnucleosides
lymphoid specific
Deoxycytidine kinase (dCK)
Broad substrate specificity
dAdo, dGuo, dCyd
Analogues of deoxynucleotides: antileukemic agents
purine and pyrimidineanalogues
dCyd dCMP
ATP ADP
Substrates:
dThd dTMP
ATP ADP
Thymidine kinase (dTK)
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Purine analogues
6-mercaptopurine 8-azaguanine2-chloro-2’-deoxyadenosine
(CdA, Cladribine®)
Hairy cell leukemia (HCL)
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Pyrimidine analogues containing sugar derivatives
arabinosyl-cytosine(araC; for treating AML)
2’, 3’-dideoxy-cytidine 2’-deoxy-2’, 2’--difluorocytidine
(dFdC, Gemcitabine)
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S-phase dependent enzyme
Thymidine kinase (dTK) dThd dTMP
ATP ADP
Human dTK: narrow substrate specificity
Herpes Simplex Virus : HSV- dTK: wide substrate specificity
Antiviral medication (Acyclovir)Suicide gene therapy (Gancyclovir, AIDS)
“ “Salvage” reaction of Salvage” reaction of deoxydeoxynucleosidesnucleosides “ “Salvage” reaction of Salvage” reaction of deoxydeoxynucleosidesnucleosides
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Acyclovir Stavudine Didanosine
Antiviral nucleoside analogues
9-hydroxyethoxy-methyl guanine,
an acyclic nucleoside
inhibition of viral DNA (RNA) replicationherpes viruses, HIV
inhibitors of retroviralreverse transcriptase
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„de novo” synthesis of purine- pyrimidine nucleotides
„free” (no sugar) purine/pyrimidine ring containing intermedier
NO YES (orotic acid)
PRPP is an allosteric activator
YES YES
C1-TH4 donor YES NO (except thymidine!)
role of Asp N donor incorporates
origin of amino groups Asp, Gln Gln
origin of the atoms in the ring
Gly, Asp, Gln, C1-THF, CO2
AspCO2, NH3 (Gln, Glu)
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„salvage” pathway of purine- pyrimidine nucleotides
mainly from bases through PRPP reaction YES NO
PRPP is necessary YES NO
mainly from nucleosides NO YES
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Catabolic pathway of purine- pyrimidine nucleotides
N of rings will be excreted YES NO
amino groups savedYES YES
diseases hyperuricemia orotic aciduria
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